In recent years, surface-enhanced Raman scattering (SERS) labels are becoming more important as an alternative to fluorescent labels.
The effect of Raman scattering occurs when a beam of light interacts with a molecule, not an atom. Part of the incident light is reflected, and part of it is scattered. Over 99% of the scattered radiation has the same frequency as the incident beam and is called Mie and Rayleigh scattering. However, a small portion of the scattered radiation has frequencies different from that of the incident beam and is called Raman and Brilliouin scattering which forms of inelastic scattering. The frequency differences between the incident and inelastically scattered radiation are determined by the properties of the molecules of which the material under study is made and are characteristic for every molecule, like a fingerprint. The Raman scattered radiation has energies slightly less than the incident photon (Stokes shift). Those energies correspond to some of the various vibrations and/or rotations of the target molecule. The Raman spectrum for a given molecule in a given environment is always the same irrespective of the frequency of the incident light. This is in contrast to fluorescence which absorbs light when the frequency or photon energy matches the energy difference between two energy levels of the molecule.
The use of Raman Scattering to investigate molecules absorbed on surfaces was initially thought to be of insufficient sensitivity. However, it was discovered that certain molecules and metal surfaces could display Raman scattering cross-sections many orders of magnitude greater than for isolated molecules. Increases in the intensity of Raman signal have been regularly observed on the order of 104-106, and can be as high as 108 and 1014. The importance of SERS is that it is both surface selective and highly sensitive where as Raman scattering is neither. The phenomenon of SERS is generally explained by a combination of an electromagnetic (EM) mechanism describing the surface electron movement in the substrate, such as a metal particle, and a chemical mechanism related to charge transfer (CT) between the substrate and a Raman active molecule.
For the chemical enhancement process, it is thought that the metal of the metal particle aids in CT excitations between the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO) of the adsorbate, i.e. the molecule bound to the surface. These excitations are possible if the Fermi level of the metal is approximately halfway between the HOMO and LUMO of the adsorbate, which in turn allows CT processes to occur at approximately half the energy of the inherent intramolecular excitations. Naturally, this effect varies from molecule to molecule, but because the energetically lowest-lying CT process is in the near ultraviolet for most organic molecules, this metal-aided process occurs in the visible spectrum.
Known nanometer-sized SERS labels usually consist of a coating layer of Raman reporter molecules on the surface of metal nanoparticles (metal NPs) which act as the source of enhancement. Compared with fluorescent labels, Raman reporters on metal NPs are resistant to photo-bleaching owing to the quenching of fluorescence excited states by metal surface and to the short lifetime of Raman virtual energy states. In addition, multiple reporters can be excited with a single light source of choice (such as near infrared light for human tissue), giving multiple sets of narrow peaks characteristic of the individual reporters. They might therefore be usable for applications in various biomedical systems.
Direct attachment of Raman reporters to metal NPs is a known technique. However, the efficiency and reliability of the nanoprobes are often compromised by ligand dissociation or exchange and the exposed Raman reporters would be easily influenced by variations in chemical or biological environments. A variety of encapsulation methods were, therefore, developed to enhance the stability of the nanoprobes, by coating them with biomolecules (such as bovine serum albumin), PEG-SH, or inorganic layers (such as SiO2).
While all these approaches reduce the dissociation of Raman reporters and provide secure anchoring points for labelling, the silica-coated nanoprobes were shown to be superior as they were impermeable to dye molecules, remarkably stable in salt media and in organic solvents, and their signals unaffected by the attachment of biomolecules (Mulvaney, S. P., Musick, M. D., et al., 2003, Langmuir, vol. 19, pp. 4784). Unlike surface-adsorbed biomolecules or ligands, the SiO2 shell is chemically stable and does not dissociate. More importantly, a well-defined core/shell structure provides unambiguous composition of the nanoprobes with minimal overall diameter, as opposed to NP aggregates with nonspecific sizes.
The growth of SiO2 shell on metal NPs typically require two types of ligands, one as the Raman reporter and one to make the metal surface amenable to SiO2 attachment. This requirement leads to multiple problems in designing SERS labels. The vitreophilic (glass loving) molecules are typically NH2- or SH-ended silanes while the Raman reporters typically have aromatic groups. These two types of ligands do not naturally mix and there is no known Raman reporter that is vitrophilic. So far, only limited number of reporters has been shown to be compatible with the SiO2 coating. Segregation of the two types of ligands could occur and lead to non-uniform distribution of the reporter molecules among the individual NPs. Furthermore, only a small fraction of the metal surface are available for Raman reporter molecules, or otherwise SiO2 would not be able to form a continuous shell on the metal NPs. The low surface concentration of Raman reporter inevitably leads to weak SERS signals. The growth of SiO2 layer is another source of problems. Since it is kinetically controlled, the shell thickness depends on the growth conditions and is not intrinsically uniform. The growth conditions need to be precisely controlled and it was also required to avoid the formation of pure silica particles and the aggregation of the NPs. Typical syntheses in this system requires more than 30 h of complicated procedures such as dialysis and ion-exchange.
Therefore, it is an object of the present invention to provide alternative Raman active labels which overcome some of the above problems.